*Target (protein/gene name): Protein Phosphatase 2A, Putative *NCBI Gene # or RefSeq#:3656063 *Protein ID (NP or XP #) or Wolbachia#:XP_843718.1 *Organism (including strain): Trypanosoma brucei brucei (TREU927) Etiologic Risk Group (see link below): 2 */Disease Information (sort of like the Intro to your Mini Research Write up):
African Trypanosomiasis, colloquially known as the sleeping sickness, is a potentially fatal disease occurring in 36 sub-Sahara African nations. In 2012 6,314 new cases of Trypanosomiasis were reported to the WHO, although it is estimated that over 20,000 new cases evolved in 2012 and 65 million people are currently at risk for contracting the disease. Trypanosomiasis is caused by the infection of protozoan parasites: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. T. b. gambiense accounts for 98% of human infections, whereas T. b. rhodesiense is accounts for only 2% but is faster acting. Both Species are transmitted to humans via bites from the Tsetse fly of the genus Glossina. Sleeping sickness is categorized into two stages. During the haemo-lymphatic stage T. brucei multiplies in subcutaneous tissue. This first stage is characterized by minimal symptoms such as fever and itching or is asymptomatic. The meningo-encephalic stage is the final stage in which T. brucei invades the central nervous system resulting in sensory and sleep cycle disturbances. If left untreated, Trypanosomiasis is fatal. Link to TDR Targets page (if present): http://www.tdrtargets.org/published/browse/t/91 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/protein/XP_843718.1 Essentiality of this protein: significant loss of fitness in bloodstream form (3 days) Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimeric state 8 Complex of proteins?: No Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Druggable in Human, 2NPP chain F with Microsystine http://dx.doi.org/10.1016/j.cell.2006.11.033
Figure 1: Reaction catalyzed by phosphoprotein phosphatase (3.1.3.16)
Enzyme Assay Information:
Colorimetric assay of protein phosphatases using p-Nitrophenylphosphate
Colorimetric assay of protein phosphatases using a synthetic phospho-peptide substrate and malachite green reagent
Assay of protein phosphatases using 32P-labeled substrates
Link to paper with assay information, required reagents, and step-by-step protocols: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965627/ Costs, quantities of substrate reagents, suppliers, and catalog #'s for the colorimetric assay of protein phosphatases using p-Nitrophenylphosphate
10 mM pNPP solution (solution can be made from tablets diluted with water). pNPP is sold in packages of 5 tablets for $34.10 per package. The supplier is Sigma-Aldrich and the catalog number is N1891-5SET.
1x calorimetric buffer (see website above for full ingredients list).
5 N NaOH. 50% sodium hydroxide solution is sold in bottles of 25 ml for $26.70 per bottle. The supplier is Sigma-Aldrich and the catalog number is 415413-25ML.
Structure:
Homology Model: PDB# 2YNL of Human PP2A.
Pairwise Alignment:
Figure 1- Blast pairwise alignment of Trypanosome brucei PP2A to the C Chain Homology Model of Human PP2A (PDB# 2NYL)
Query Coverage: 476
Max Identities: 71%
% Positives: 84%
Chain used for Homology: C Current Inhibitors: Okadaic Acid and Microcystin: Have been used to discover the function of PP2A in cells, but are too toxic to be used as drugs Fostriecim: Causes apoptosis of cells by inducing early mitosis; is highly susceptible to oxidation and Phase I trials were cancelled; is currently being tested for ways to prevent its oxidation Canthardin/Norcanthardin: Canthardin is a strong inhibitor with an IC50 of 0.194 and it's able to transverse membranes; its analog Norcanthardin is currently being tested as a drug candidate because Canthardin has high toxicity levels Expression Information:
Expressed in E. coli cells: http://www.sciencedirect.com/science/article/pii/S0378111901003675 Purification Method:
Similar Protein PP5 was purified with Ni-NTA chromatography using 6His tagged proteins: http://www.sciencedirect.com/science/article/pii/S0378111901003675 PyMol Image of Protein:
Figure 2- PyMol image of Human PP2A (PDB 2NYL) homolog. Protein is shown as a surface (60% transparency) with the A and B regulatory subunits colored where Carbon is green and orange, respectively (dark blue is Nitrogen, red is Oxygen). C catalytic subunit colored where Carbon is light blue. Known inhibitor Microcystin-LR is shown as sticks and colored where Carbon is pink. Cofactor Manganese-II ions are shown as spheres and colored grey.
Figure 3- PyMol image of Human PP2A (PDB 2NYL) homolog active site with known inhibitor Microcystin-LR docked. Catalytic subunit C shown as surface (60% transparency) and colored where Carbon is light blue. Microcystin-LR is shown as sticks and colored where Carbon is pink. Cofactor Manganese-II ions are shown as spheres and colored grey.
Molar Extinction coefficient at 280 nm wavelength: Ext. coefficient 43360 Abs 0.1% (=1 g/l) 1.197, assuming that all pairs of Cys residues form cystines
Ext. coefficient 42860 Abs 0.1% (=1 g/l) 1.183, assuming that all Cys residues are reduced
TMpred graph:
:
Most likely no transmembrane proteins; no possible models found.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): ATGCCGGCGACCACCTCTATCCCGCCTGTGACTGCGCCTGGTACCGGTGGTGAGGCGTCTTCCTTCAATGTTGACGAGCTCATCCAGTACGTTCTGCAGGGTAAACCGCTGTCTGAACCGCAGGTTGCCCGTCTGTGCCAGAAAGCGCGTGAAGTTCTGGAAAAAGAAGAAAACGTGCACACCGTTCGTGCGCCAGTTACCGTTTGCGGTGACATCCATGGTCAGTTCCACGATCTGCTGGAACTCCTGAAAATTGGTGGTCTCCCACCGGACACCAACTATCTCTTCATGGGTGACTACGTTGATCGCGGCTACTACTCCGTTGAAACCGTTACGCTCCTCCTGCTGTTCAAAATCCGCTACCCGGAGCGTGTTCAGATCCTGCGTGGTAATCATGAATCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGCGTTCGTAAATACGGTTCTGCGAATGTTTGGAAACTGCTGACGGATCTGTTCGACTACCTGCCGCTGGCGGCTGTTGTTGAAAACGAAATCTTCTGCCTGCACGGTGGCCTGTCTCCGACCCTGGACTCTCTGGCTCACATCCGTTCTCTGGAACGTGTCCAAGAGGTCCCGCATGAAGGCCCGATGTGCGACCTCCTCTGGTCCGATCCGGAAGACAAAGACGGTTGGGGTATCTCTCCGCGTGGTGCGGGTTTCACCTTCGGTGCGGACATTACCGAACAGTTCTGCCACAACAATGGTCTGAAAACCATCGCGCGTGCTCACCAGCTCGTAGCCGAGGGTTACTCTTGGGCGCACTCTGATAAACTGGTTACTATCTTCTCTGCGCCGAACTACTGCTATCGTTGTGGTAACCTGGCGGGTCTGCTCGAACTCGACGAACACATGAACAAATGCTTCTTTCAGTTCGACCCGGCACCTCGTCGCGGTGAAGCACAAGTCTCCAAGAAGACCCCGGACTACTTCCTGTAA *GC% Content for gene (codon optimized): 54.7%
*NCBI Gene # or RefSeq#: 3656063
*Protein ID (NP or XP #) or Wolbachia#: XP_843718.1
*Organism (including strain): Trypanosoma brucei brucei (TREU927)
Etiologic Risk Group (see link below): 2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
African Trypanosomiasis, colloquially known as the sleeping sickness, is a potentially fatal disease occurring in 36 sub-Sahara African nations. In 2012 6,314 new cases of Trypanosomiasis were reported to the WHO, although it is estimated that over 20,000 new cases evolved in 2012 and 65 million people are currently at risk for contracting the disease. Trypanosomiasis is caused by the infection of protozoan parasites: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. T. b. gambiense accounts for 98% of human infections, whereas T. b. rhodesiense is accounts for only 2% but is faster acting. Both Species are transmitted to humans via bites from the Tsetse fly of the genus Glossina. Sleeping sickness is categorized into two stages. During the haemo-lymphatic stage T. brucei multiplies in subcutaneous tissue. This first stage is characterized by minimal symptoms such as fever and itching or is asymptomatic. The meningo-encephalic stage is the final stage in which T. brucei invades the central nervous system resulting in sensory and sleep cycle disturbances. If left untreated, Trypanosomiasis is fatal.
Link to TDR Targets page (if present):
http://www.tdrtargets.org/published/browse/t/91
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/protein/XP_843718.1
Essentiality of this protein: significant loss of fitness in bloodstream form (3 days)
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimeric state 8
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Druggable in Human, 2NPP chain F with Microsystine
http://dx.doi.org/10.1016/j.cell.2006.11.033
E.C. #: 3.1.3.16
Link to BRENDA E.C. # page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.16
BRENDA enzyme mechanism schematic:
Enzyme Assay Information:
- Colorimetric assay of protein phosphatases using p-Nitrophenylphosphate
- Colorimetric assay of protein phosphatases using a synthetic phospho-peptide substrate and malachite green reagent
- Assay of protein phosphatases using 32P-labeled substrates
Link to paper with assay information, required reagents, and step-by-step protocols:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965627/
Costs, quantities of substrate reagents, suppliers, and catalog #'s for the colorimetric assay of protein phosphatases using p-Nitrophenylphosphate
Structure:
Homology Model: PDB# 2YNL of Human PP2A.
Pairwise Alignment:
Query Coverage: 476
Max Identities: 71%
% Positives: 84%
Chain used for Homology: C
Current Inhibitors:
Okadaic Acid and Microcystin: Have been used to discover the function of PP2A in cells, but are too toxic to be used as drugs
Fostriecim: Causes apoptosis of cells by inducing early mitosis; is highly susceptible to oxidation and Phase I trials were cancelled; is currently being tested for ways to prevent its oxidation
Canthardin/Norcanthardin: Canthardin is a strong inhibitor with an IC50 of 0.194 and it's able to transverse membranes; its analog Norcanthardin is currently being tested as a drug candidate because Canthardin has high toxicity levels
Expression Information:
Expressed in E. coli cells: http://www.sciencedirect.com/science/article/pii/S0378111901003675
Purification Method:
Similar Protein PP5 was purified with Ni-NTA chromatography using 6His tagged proteins: http://www.sciencedirect.com/science/article/pii/S0378111901003675
PyMol Image of Protein:
Amino Acid Sequence:
MPATTSIPPVTAPGTGGEASSFNVDELIQYVLQGKPLSEPQVARLCQKAREVLEKEENVHTVRAPVTVCGDIHGQFHDLLELLKIGGLPPDTNYLFMGDYVDRGYYSVETVTLLLLFKIRYPERVQILRGNHESRQITQVYGFYDECVRKYGSANVWKLLTDLFDYLPLAAVVENEIFCLHGGLSPTLDSLAHIRSLERVQEVPHEGPMCDLLWSDPEDKDGWGISPRGAGFTFGADITEQFCHNNGLKTIARAHQLVAEGYSWAHSDKLVTIFSAPNYCYRCGNLAGLLELDEHMNKCFFQFDPAPRRGEAQVSKKTPDYFL
Length of protein in Amino Acids: 323 aa
Molecular Weight of protein in (kD): 36,243.1
Molar Extinction coefficient at 280 nm wavelength:
Ext. coefficient 43360 Abs 0.1% (=1 g/l) 1.197, assuming that all pairs of Cys residues form cystines
Ext. coefficient 42860 Abs 0.1% (=1 g/l) 1.183, assuming that all Cys residues are reduced
TMpred graph:
:
Most likely no transmembrane proteins; no possible models found.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCCGGCGACCACCTCTATCCCGCCTGTGACTGCGCCTGGTACCGGTGGTGAGGCGTCTTCCTTCAATGTTGACGAGCTCATCCAGTACGTTCTGCAGGGTAAACCGCTGTCTGAACCGCAGGTTGCCCGTCTGTGCCAGAAAGCGCGTGAAGTTCTGGAAAAAGAAGAAAACGTGCACACCGTTCGTGCGCCAGTTACCGTTTGCGGTGACATCCATGGTCAGTTCCACGATCTGCTGGAACTCCTGAAAATTGGTGGTCTCCCACCGGACACCAACTATCTCTTCATGGGTGACTACGTTGATCGCGGCTACTACTCCGTTGAAACCGTTACGCTCCTCCTGCTGTTCAAAATCCGCTACCCGGAGCGTGTTCAGATCCTGCGTGGTAATCATGAATCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGCGTTCGTAAATACGGTTCTGCGAATGTTTGGAAACTGCTGACGGATCTGTTCGACTACCTGCCGCTGGCGGCTGTTGTTGAAAACGAAATCTTCTGCCTGCACGGTGGCCTGTCTCCGACCCTGGACTCTCTGGCTCACATCCGTTCTCTGGAACGTGTCCAAGAGGTCCCGCATGAAGGCCCGATGTGCGACCTCCTCTGGTCCGATCCGGAAGACAAAGACGGTTGGGGTATCTCTCCGCGTGGTGCGGGTTTCACCTTCGGTGCGGACATTACCGAACAGTTCTGCCACAACAATGGTCTGAAAACCATCGCGCGTGCTCACCAGCTCGTAGCCGAGGGTTACTCTTGGGCGCACTCTGATAAACTGGTTACTATCTTCTCTGCGCCGAACTACTGCTATCGTTGTGGTAACCTGGCGGGTCTGCTCGAACTCGACGAACACATGAACAAATGCTTCTTTCAGTTCGACCCGGCACCTCGTCGCGGTGAAGCACAAGTCTCCAAGAAGACCCCGGACTACTTCCTGTAA
*GC% Content for gene (codon optimized): 54.7%